集落刺激因子1受体抑制剂培西达替尼对脂多糖刺激骨髓来源巨噬细胞衰老的调节作用  

Regulation of colony-stimulating factor 1 receptor inhibitor pexidartinib on the senescence of mouse bone marrow-derived macrophages stimulated by lipopolysaccharide

作　　者：肖天骄 张洁 康佳兵 李丽 湛济帆 韦艳 田艾 Xiao Tianjiao;Zhang Jie;Kang Jiabing;Li Li;Zhan Jifan;Wei Yan;Tian Ai(School of Stomatology,Guizhou Medical University,Guiyang 550004,China;Department of Stomatology,The First Affiliated Hospital of Guiyang University of Traditional Chinese Medicine,Guiyang 550004,China;Department of Prosthodontics and Implant Dentistry,School and Hospital of Stomatology,Guizhou Medical University,Guiyang 550004,China)

机构地区：[1]贵州医科大学口腔医学院,贵阳550004 [2]贵阳中医药大学第一附属医院口腔科,贵阳550004 [3]贵州医科大学口腔医学院·附属口腔医院口腔修复种植科,贵阳550004

出　　处：《中华口腔医学杂志》2023年第6期575-583,共9页Chinese Journal of Stomatology

基　　金：国家自然科学基金(82260193,81760192)。

摘　　要：目的探讨集落刺激因子1受体(colony-stimulating factor 1 receptor,CSF-1R)抑制剂培西达替尼(pexidartinib,PLX3397)对脂多糖(lipopolysaccharide,LPS)刺激下的骨髓来源巨噬细胞(bone marrow-derived macrophages,BMDM)衰老的调节作用。方法从10只6~8周龄雄性C57BL/6小鼠(贵州医科大学实验动物中心获取)的股骨和胫骨分离、培养BMDM,将BMDM分为空白对照组、LPS组(1μg/ml LPS处理24 h)和低、中、高浓度PLX3397预处理组(分别给予100、500和1000 nmol/L PLX3397处理4 h后,再以1μg/ml LPS处理24 h),采用细胞计数(cell counting kit-8,CCK-8)试剂盒检测细胞活力;通过衰老相关β-半乳糖苷酶(senescence-associated-β-galactosidase,SA-β-gal)染色检测细胞衰老;蛋白质印迹法检测细胞周期依赖性激酶抑制因子p16、p21和CSF-1R的蛋白表达;细胞免疫荧光法检测p16、p21在细胞内的表达;实时定量PCR(real-time fluorescence quantitative PCR,RT-qPCR)检测衰老相关分泌表型(senescence-associated secretory phenotype,SASP)因子包括白细胞介素(interleukin,IL)、趋化因子1/10(chemokine-1/10,CXCL-1/10)、基质金属蛋白酶8(matrix metalloproteinase-8,MMP-8)、转化生长因子-β(transforming growth factor-β,TGF-β)的mRNA表达。结果中、高浓度PLX3397预处理组SA-β-gal染色阳性细胞率[分别为(39.33±4.93)%、(36.33±3.06)%]均较LPS组[(52.00±3.00)%]显著下调(P=0.020,P=0.005);低、中、高浓度PLX3397预处理组CSF-1R的蛋白表达(分别为0.74±0.18、0.61±0.07、0.54±0.06)均较LPS组(1.16±0.08)显著下调(P=0.013,P=0.002,P<0.001);低、中、高浓度PLX3397预处理组CSF-1R的mRNA表达(分别为1.04±0.06、0.90±0.05、1.18±0.08)均较LPS组(2.90±0.25)显著下调(P<0.001);低、中、高浓度PLX3397预处理组p16平均荧光强度(分别为49.76±3.65、48.21±1.72、47.99±1.26)均显著低于LPS组(66.88±5.85)(P=0.001,P<0.001,P<0.001);中、高浓度PLX3397预处理组p21平均荧光强度(分别为34.43±3.62、30.13±0.86)均较LPS组(Objective To investigate the effects of colony-stimulating factor 1 receptor(CSF-1R)inhibitor pexidartinib(PLX3397)on the senescence of bone marrow-derived macrophages(BMDM)stimulated by lipopolysaccharide(LPS).Methods BMDM were isolated and cultured from femurs and tibiae of 10 male C57BL/6 mice aged 6-8 weeks(obtained from Laboratory Animal Center of Guizhou Medical University).They were divided into blank control group,LPS group(treated with 1μg/ml LPS for 24 h)as well as low,medium and high concentration PLX3397 pretreatment groups(treated with 100,500 and 1000 nmol/L PLX3397 for 4 h respectively followed by 1μg/ml LPS for 24 h).The corresponding markers of macrophages were detected by flow cytometry.Cell viability was detected by cell counting kit-8 and cellular senescence was detected by senescence-associated-β-galactosidase(SA-β-gal)staining.Meanwhile,protein expressions of cycle-dependent kinase inhibitor p16,p21 and CSF-1R were detected by Western blotting,and the expressions of p16 and p21 were detected by intracellular immunofluorescence.Real-time fluorescence quantitative PCR(RT-qPCR)was used to investigate the mRNA levels of senescence-associated secretory phenotype(SASP)genes including interleukin(IL),IL-1β,chemokine-1/10(CXCL-1/10),matrix metalloproteinase-8(MMP-8),and transforming growth factor-β(TGF-β).Results The rate of SA-β-gal positive staining in medium and high concentration PLX3397 pretreatment groups[(39.33±4.93)%and(36.33±3.06)%respectively]were significantly downregulated compared with LPS group[(52.00±3.00)%](P=0.020,P=0.005).The expression of CSF-1R protein in low,medium and high concentration PLX3397 pretreatment groups were(0.74±0.18,0.61±0.07,0.54±0.06),all of which were significantly lower than that in LPS group(1.16±0.08)(P=0.013,P=0.002,P<0.001).The expression levels of CSF-1R mRNA in low,medium and high concentration PLX3397 pretreatment groups(1.04±0.06,0.90±0.05,1.18±0.08)showed similar trend(2.90±0.25)(P<0.001).The average fluorescence intensity of p16 in

关 键 词：细胞衰老 受体 巨噬细胞集落刺激因子 脂多糖类 骨髓来源巨噬菌细胞 

分 类 号：R965[医药卫生—药理学]